Rice seeds contain abundant starch and have been commonly used in the food and beverage industries. Generally, rice seed contains 6-10% of protein and 70-80% of starch of total seed weight, and the protein and starch can be separated for processing into different products. The traditional process for separating rice protein from starch can be tedious and costly, while the use of chemicals, e.g., sodium hydroxide, acids, and surfactants, is undesirable in food production. As an alternative, an enzymatic process can produce high-maltose syrup and high-protein rice flour from milled rice (Shaw and Sheu, 1992, Biosci. Biotech. Biochem. 56:1071-1073). In this process, the rice flour is first liquefied with thermostable microbial xcex1-amylase at high temperature and the heat-coagulated protein is separated from the soluble starch hydrolysate and recovered as high-protein rice flour. The starch hydrolysate is further treated with microbial xcex2-amylase and debranching enzyme (isoamylase and/or pullulanase) to produce high-maltose syrup. The syrup can be used for food processing and alcohol beverage production. The high-protein rice flour has high nutritional value and is useful for the production of pudding, gruel, instant milk, baby food, etc.
The development of other alternative methods to facilitate utilization of cereal seed starch is desirable.
The invention is based, in part, on the inventor""s surprising discovery that a microbial amylopullulanase (APU), e.g., Thermoanaerobacter ethanolicus APU, e.g., a truncated T. ethanolicus APU, when expressed under the control of a seed specific promoter in a seed, e.g., a germinated seed, shows a specific activity several-fold higher than when expressed in E. coli. Thus, a system has been developed and is described herein, whereby T. ethanolicus APU, e.g., a truncated T. ethanolicus APU, e.g., a T. ethanolicus APU lacking amino acids 1-105 and 1061-1481 of the mature APU (SEQ ID NO:1), is expressed in a seed (e.g., a rice seed), thereby producing a seed with an altered starch or protein content. Such seeds can be used in the production of plant starches or sugars beneficial to numerous industries, including the cereal and beverage production industries.
When a T. ethanolicus APU sequence is said to be free of amino acids 1-105 and 1061-1481 of SEQ ID NO:1, it means that the APU sequence does not contain the complete sequence defined by amino acids 1-105 and 1061-1481 of SEQ ID NO:1. Thus, the APU sequence can contain a portion of amino acids 1-105 and 1061-1481 of SEQ ID NO:1 and still be considered free of amino acids 1-105 and 1061-1481 of SEQ ID NO:1. Suitable truncated APU sequences for use in the constructs described herein can even contain all but one, 25, 50, 100, 150, 200, 300, 400, 500, or more amino acids defined by the sequences of 1-105 and 1061-1481 of SEQ ID NO:1 and still be considered free of SEQ ID NO:1.
Accordingly, in one aspect, the present invention features a DNA construct that includes a nucleotide sequence encoding a microbial amylopullulanase or a fragment thereof having pullulanase and xcex1-amylase activities, operably linked to a seed-specific promoter. The microbial amylopullulanase can be T. ethanolicus Amylopullulanase, e.g., T. ethanolicus 39E Amylopullulanase. In one aspect, a truncated T. ethanolicus 39E Amylopullulanase that retains both xcex1-amylase and pullulanase activities is used, e.g., the construct includes a nucleotide sequence encoding a truncated T. ethanolicus 39E Amylopullulanase that is free of amino acids 1-105 and 1061-1481 of SEQ ID NO:1. The construct can also include a sequence encoding a signal peptide, e.g., a glutelin signal peptide, upstream of the Amylopullulanase coding sequence. In addition, the construct can include a 3xe2x80x2 gene terminator sequence, e.g., a nopaline synthase gene terminator sequence. The seed specific promoter of the construct can be any plant promoter that is expressed in seeds, preferably in germinating or developing seeds. Exemplary seed specific promoters include a glutelin promoter, e.g., the GluB promoter and an xcex1-Amy promoter, e.g., xcex1-Amy3 or xcex1Amy8 promoters.
In another aspect, the invention features a genetically engineered seed, e.g., a rice, corn, wheat, or barley seed, that includes a DNA construct having a nucleotide sequence encoding a microbial amylopullulanase enzyme or a fragment thereof having pullulanase and xcex1-amylase activities, operably linked to a seed-specific promoter, e.g., a DNA construct described hereinabove. Such seeds can have a modified starch structure or content, including reduced amylose content or altered total starch composition compared to naturally occurring seeds. Such seeds can thus be the source of sugars and high protein seed products.
In yet another aspect, the invention features a method of producing a starch having a modified structure. The method includes the steps of: (a) transforming a plant cell with a DNA construct that includes a nucleotide sequence encoding a microbial amylopullulanase or a fragment thereof having pullulanase and xcex1-amylase activities, operably linked to a seed-specific promoter, e.g., a DNA construct described hereinabove; (b) generating a whole plant from the transformed plant cell; (c) optionally multiplying the whole plant; (d) harvesting seeds from the whole plant or multiplied whole plants; and (e) extracting the starch from the seeds. The seed can be a rice, corn, wheat, or barley seed. In a preferred embodiment, the seed is a rice seed.
In another aspect, the invention features a method of producing a sugar. The method includes: (a) transforming a plant cell with a DNA construct comprising a seed specific promoter operatively linked to a nucleotide sequence encoding a microbial amylopullulanase or a fragment thereof having pullulanase and xcex1-amylase activities, e.g., a DNA construct described herein; (b) generating a whole plant from the transformed plant cell; (c) optionally multiplying the whole plant; (d) harvesting seeds from the whole plant or multiplied whole plants; and (e) treating the seeds, or starch extracted from the seeds, under conditions sufficient to convert the starch in the seeds or the starch extracted from the seeds, to sugar. In one embodiment, the seed is a rice seed. An exemplary manner of treating the seeds, or starch extracted from the seeds, includes heating the seeds, or starch extracted from the seeds, until the starch turns to sugar. For example, the seeds or starch can be heated to between about 60 to 95xc2x0 C., e.g., at least about 60xc2x0 C., 70xc2x0 C., 75xc2x0 C., 80xc2x0 C., 85xc2x0 C., 90xc2x0 C., 95xc2x0 C.
In yet another aspect, the invention features a method of making a polypeptide. The method includes: providing a nucleic acid construct that includes a glutelin promoter, e.g., a GluB promoter, operatively linked to a nucleic acid sequence encoding a heterologous polypeptide, e.g., an enzyme or functional fragment thereof, e.g., a bacterial enzyme or functional fragment thereof; introducing the nucleic acid construct into a cell, e.g. a plant cell, e.g., a rice cell; and allowing the cell to express the polypeptide encoded by the coding sequence. The sequence encoding the heterologous polypeptide optionally includes a signal sequence, e.g., a glutelin signal sequence. The cell can be a tissue culture cell. In one embodiment, the cell is a seed cell and the polypeptide is expressed in the endosperm of a germinating seed. In another embodiment, the cell is a seed cell and the polypeptide is expressed in the embryo of a developing seed. In another embodiment, the cell is a tissue culture cell and the polypeptide is secreted into the culture medium of the cell.
A xe2x80x9cDNA constructxe2x80x9d is defined herein as a DNA molecule that has been modified to contain segments of DNA that are combined and juxtaposed in a manner that would not otherwise exist in nature. The term encompasses plasmid and viral constructs.